Given the capability of modulating individual gene expression in tumor cells, RNA agents such as small interfering RNA (siRNA) and messenger RNA (mRNA) have demonstrated tremendous potential in revealing the functionality of specific gene alterations and enabling novel classes of therapies for cancer treatment. The effective systemic RNA delivery to tumors, however, remains a formidable challenge for the widespread application of RNA technologies in cancer research and therapy. In this project, we propose to (i) develop robust nanoparticle (NP) platforms for effective in vivo delivery of siRNA and mRNA to tumor tissue, and (ii) apply this technology to relevant cancer therapeutic targets for treating non-small cell lung cancer (NSCLC). In recent efforts, we have conceived and developed a new generation of lipid-polymer hybrid NPs with promising features for systemic siRNA delivery, including long blood circulation, high tumor accumulation, and impressive gene silencing efficacy. We have also successfully employed these hybrid NPs to explore a putative therapeutic target, Prohibitin1 (PHB1), in NSCLC and to deliver tumor suppressor-encoded mRNA to cancer cells, as evidenced by inhibition of tumor cell growth in vitro and following systemic delivery in vivo. In light of these extensive and encouraging preliminary results, we hypothesize that the new lipid-polymer hybrid NP-mediated systemic RNA delivery will become a useful platform for in vivo evaluation of cancer targets and for the development of novel RNA therapies for cancer treatment. In Aim 1, we will systematically optimize, understand and evaluate specific parameters that have been shown to control gene silencing efficiency and in vivo properties (e.g., pharmacokinetics, biodistribution, and side effects) of the hybrid NPs. In Aim 2, we propose to explore the mechanisms and pathways underlying the PHB1 silencing-induced anti-tumor effect, using the optimized siRNA NPs from Aim 1. The hybrid NPs for systemic delivery of anti-PHB1 siRNA will be extensively examined in multiple NSCLC xenograft and orthotopic models. In Aim 3, we will expand and refine the new hybrid NPs to deliver mRNA coding for tumor suppressors relevant to NSCLC, including PTEN and LKB1. This mRNA delivery approach for cancer therapy will be systematically investigated in vitro and in NSCLC xenograft and genetically engineered mouse models. At the conclusion of this project, we expect that the convergence of innovative RNA delivery strategy and significant discoveries in tumor biology will further strengthen our arsenal of therapies against NSCLC and other aggressive cancers.